Pichia pastoris mutant

Abstract

It refers to a Pichia pastoris mutant. It refers also to a method to obtain the mutant, to a method to obtain protein-glutamine glutaminase (PGG), a new protein-glutamine glutaminase (PGG), and to the use of said mutant for obtaining new PGG and PPG.

Description

[0001]

[0002] Pichia pastoris mutant”

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to the field of genetic engineering, in particular to a Pichia pastoris mutant and its use for the production of protein-glutamin glutaminase.

[0005] BACKGROUND ART

[0006] Many plant proteins contain amide groups or glutamine and asparagine residues, which are cross-linked with other amino acids, which impair the solubility, the emulsification capacity, the foaming, and the gelation capacity, critical properties of food proteins, limiting their application in the food industry, as disclosed in Lura et al., Role of Peptide Conformation in the Rate and Mechanism of Deamidation of Asparaginyl Residues, Biochem., 1988, 27, 7671-7677, and Zhang, etal., Protein-glutaminase: Research progress and prospect in food manufacturing, Food Bioscience, 2021, 43, 101314.

[0007] In prior art, it is disclosed the development of chemical and physical approaches for protein deamidation, which are efficiently used to improve protein functionalities through modification of the amide group in the side chain of the amino acids asparagine or glutamine, as disclosed in Zink et al., Physical, Chemical and Biochemical Modifications of Protein-Based Films and Coatings: An Extensive Review, Int. J. Mol. Sci., 2016, 17(9), 1376.

[0008] Protein deamidation mainly occurs through the transformation of the amide side chains of asparagine and glutamine into negatively charged carboxyl groups via the release of ammonia, to stretch the structure of the protein and improve the solubility of the protein, as disclosed in Chen et al., Protein deamidation to produce processable ingredients and engineered colloids for emerging food applications, Compr. Rev. Food Sci. Food Saf., 2021,20(4), 3788-3817.

[0009] While such chemical and physical deamidation processes improve the quality of proteins, side effects, such as peptide hydrolysis, may be present.

[0010] Therefore, enzymatic deamidation is preferred for food systems because of its high specificity, mild reaction conditions, and safe modification.

[0011] Specifically, protein-glutamin glutaminase (PGG; EC 3.5.1.44) is efficient in hydrolysing the amide group of glutamine residues of proteins, which improves the quality of food proteins that exhibit low water solubility. Said enzyme was initially isolated from the soil bacteria Chryseobacterium proteolyticum, as disclosed in Yamaguchi et al., Eur. J. Biochem., 2001, 268, 1410-1421.PGG (Protein-Glutamine Glutaminase) is suitable to be used in a broad range of applications, such as the deamidation of corn protein, soybean protein, casein, wheat gluten, skimmed milk, whey protein, yogurt, oat protein, among others.

[0012] Further, PGG has no protease or glutamine transaminase activity and can be used to reduce the allergenicity of food proteins by decreasing the proportion of glutamine, as disclosed in Tanabe et al., A Major Wheat Allergen Has a Gln-GIn-GIn-Pro-Pro Motif Identified as an IgE-Binding Epitope, Biochem. Biophys. Res. Comm., 1996, 219, 290-293.

[0013] According to Liu et al., Application Prospect of Protein-Glutaminase in the Development of Plant-Based Protein Foods, Foods, 2022, 11 , 440, enzymatic deamidation by protein-glutaminase can enhance the physical and chemical properties of plant-based proteins, which may open opportunities for researchers to design plant-based protein foods which are comparable to animal counterparts in appearance, texture, and flavour, providing broad application prospects for PGG in the food industry.

[0014] However, the wild-type strain of C. proteolyticum is characterized by low production of PGG and makes unsuitable for industrial applications.

[0015] In prior art were disclosed different approaches to develop efficient strategies to improve the PGG production for large-scale industrial applications.

[0016] One of the approaches is addressed to heterologous expression systems for PPG production.

[0017] Although, genetic engineered strains of Escherichia coli, as disclosed in, for example, Li et al., Recombinant expression of protein-glutaminase and optimization of fermentation conditions, Food Ferment. Ind., 2021, 4(3) 294-301, and Corynebacterium glutamicum, as disclosed in, for example, Kikuchi et al., Production of Chryseobacterium proteolyticum protein-glutaminase using the twin-arginine translocation pathway in Corynebacterium glutamicum, 2007, 78, 67-74, have been developed to overexpress PGG and increase the production thereof, they still cannot reach the level of industrial production. Moreover, this approach has disadvantages. The use of E. coli is not of practical choice to produce food enzymes due to the presence of endotoxins. The use of C. glutamicum requires the use of a high-cost protease for activation, which restricts the large-scale production of PGG.

[0018] By optimized combination of promoter, signal peptide, and culture medium, genetic engineered strains of Bacillus subtilis and Bacillus licheniformis provided high yields of PGG, as disclosed in Niu et al., Twin-arginine signal peptide of Bacillus licheniformis GlmU efficiently mediated secretory expression of protein glutaminase, Electron. J. Biotechnol., 2019, 42, 49-55, and Yin et al., Combinatorial engineering for efficient production of protein-glutaminase in Bacillus subtilis, Enz. Microb. Technol., 2021, 150, 109863, respectively.

[0019] Another approach is the use of yeasts as host for recombinant protein expression, as disclosed in Spohner et al., Expression of enzymes for the usage in food and feed industry with Pichia pastoris, J. Biotechnol., 2015, 202, 118-134. In this review it is disclosed that the yeastSaccharomyces cerevisiae has the capability to secrete the authentic protein to the medium in a soluble form and the capacity to produce large amounts of enzymes via economically attractive downstream processing without laborious purification. However, many of the secreted proteins of S. cerevisiae are not found free in the medium, but rather in the periplasmic space, what leads to problems with purification resulting in decreased product yield. It is further disclosed that methylotrophic yeast Pichia pastoris, currently reclassified as Komagataella pastoris, is an amiable candidate for the expression of recombinant proteins and discloses a large list of enzymes used in food and feed industry expressed in P. pastoris.

[0020] In Guo et al., Heterologous expression of protein glutaminase in Pichia pastoris, Food Ferment. Ind., 2023, 49(7), 26-31, it is disclosed the optimization the culture medium to produce heterologous Pro-PGG from Pichia pastoris reaching an activity of 0.878 U / rnL.

[0021] In Chinese patent application CN-A-116200280 it is disclosed a mutant strain of P. pastoris for the production of PGG (CCTCC NO: M20211205), wherein the mutant was obtained by ultraviolet mutagenesis of an engineered strain of P. pastoris carrying a recombinant plasmid expressing PGG. The enzyme activity of the fermentation supernatant from the P. pastoris mutant reached 6.0 U / rnL.

[0022] Another approach is the prepare PGG from another microorganisms. For example, in international patent application WO-A-2024 / 032886 it is disclosed the preparation of PGG from Chitinophaga sp. using E. coli or P. pastoris. According to this disclosure, such PGG has a faster deamidation of natural proteins, as well as a higher degree of deamidation.

[0023] Thus, in spite of approaches disclosed in the prior art, there is a need for further Pichia pastoris mutants showing improved yield by the production of PGG.

[0024] SUMMARY OF INVENTION

[0025] An aspect of the invention is a Pichia pastoris mutant.

[0026] Another aspect of the invention is a method to obtain said mutant.

[0027] Another aspect of the invention a nucleic acid construct or plasmid pBIZalpha 1.4 HisproPGG and final construct (plasmid) pBIZ1.4 His-Pro-PGG delta Zeo plasmid, which corresponds strictly to the expression cassette pBIZalpha 1.4 HisproPGG depleted from the selection marker encoded through the antibiotic resistance gene of Zeocin and the use of both in the in the obtention of the new Pichia pastoris mutant.

[0028] Another aspect of the invention is the use of said mutant for preparing a new PGG (His-Pro-PGG).

[0029] Another aspect of the invention is the use of the Pichia pastoris mutant in the manufacturing of food and / or food products.

[0030] Another aspect of the invention is a new PGG, referred as His-Pro-PGG, and its use in the manufacturing of food and food products, such as plant-based beverage.DESCRIPTION OF THE FIGURES

[0031] In Figure 1 it is shown a transformed plasmid map, referred as plasmid pBIZalpha 1.4 HisproPGG map, which is characterised because it encodes the His pro PGG sequence (SEQ ID NO: 1) under A0X1 promoter and is able to transform P. pastoris. The different regions present in the transformed plasmid (recombinant plasmid), and inserted in the strain genome of the mutant yeast of the invention, as well as the size and / or the position of each of the sequence in the plasmid are shown in Table I:

[0032] TABLE I

[0033]

[0034] Each of the regions listed in table I are described below and are disclose individually and in combination with the other regions.

[0035] The pUC ori region is derived from the pUC19 plasmid, which is a commonly used vector in molecular cloning. The pPICZ plasmid is a yeast expression Pichia pastoris vector that contains the pUC ori, the replication origin, which allows the replication of the plasmid of the invention in E. coli. The pPICZ plasmid allows expressing recombinant proteins in Pichia pastoris and cloninginto Pichia pastoris cells. The pPICZ plasmid is disclosed, for example, in Cregg et al., Pichia pastoris as a host for heterologous protein expression. Mol. Biotechnol., 1993, 11, 15-71.

[0036] The noncoding region (position 675-703) is a linker region wherein restriction enzyme sites are available.

[0037] The A0X1 promoter region originates naturally from the yeast Pichia pastoris itself. It's the native promoter for the alcohol oxidase 1 (A0X1) gene within this yeast.

[0038] A0X1 promoter is a yeast endogenous promoter inducible under addition of methanol, for the expression of the target enzyme. A0X1 promoter comprise a Pmel restriction site whichenhances the recombination with the AOX promoter from the genome.

[0039] The noncoding region (position 1627-1634) is a noncoding region derived from the cloning procedures.

[0040] Kozak sequence functions as the starting point of protein translation.

[0041] Ost1-aF region is disclosed in the article Barrero et al., and it is added to the pPICZ plasmid to improved secretion signal which enhances the secretion of model proteins from Pichia pastoris and allows the transport of the protein to the extracellular region, Microb. Cell., 2018, 17, 161.

[0042] HisproPGG region is the coding region of the protein-glutaminase of interest, and it is available from UniProt (ref: Q9AQQ8), however the HisproPGG region does not comprise the signal peptide (21 aa), contained in the commercial version of PGG and instead, the HisproPGG region has been modified and comprises a His tag in N terminal in that position.

[0043] The noncoding region (position 2823-2828) is a non-coding region derived from the cloning procedures

[0044] The source of A0X1 terminator region is the p PICZ plasmid. A0X1 terminator region defines the region ending of transcription process and allow detachment of RNA polymerase from the DNA strand.

[0045] Noncoding region (position 3076-3184) is a noncoding region derived from the cloning procedures.

[0046] In Figure 2 it is disclosed vector pBGP1 Hygro CRE, which is used to remove the antibiotic resistance from the P. pastoris. Vector pBGP1 Hygro CRE encodes recombinase under constitutive expression promoter pGAP. This vector is not integrated into the genome, of P. pastoris and it remains as an episomal, due to the PARS sequence comprised in vector pBGP1 Hygro CRE, as shown in figure 2. Vector pBGP1 Hygro CRE also contains a bacterial replication origin (ori) to be able to carry out previous cloning steps in E.coli, and it also shows resistance to hygromycin and ampicillin to select clones in P. pastoris or E.coli respectively.

[0047] Figure 3 corresponds to the map of the expression cassette pBIZalpha 1.4 HisproPGG depleted from the selection marker encoded through the antibiotic resistance gene of Zeocin, referred as the final construct (plasmid) pBIZ1.4 His-Pro-PGG delta Zeo.DESCRIPTION OF THE INVENTION

[0048] The present invention relates to a Pichia pastoris mutant, reclassified as Komagataella phafifii, deposited by the company CYGYC Biocon, S.L. on May 22, 2024 under the Budapest Treaty at the Deutsche Sammlung von Mikroorganismen und Zellkulturen as the International Depository Authority (Leibniz Institut DSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, InhoffenstraBe 7 B, 38124 Braunschweig, GERMANY). The assigned deposit number was DSM 34997. For the purposes of this invention, references to Pichia pastoris are synonymous with Komagataella phaffii, consistent with the taxonomic reclassification described by Kurtzman (2009).

[0049] The inventors of the present invention have developed a new mutant of Pichia pastoris, which surprisingly exhibits a high activity in the production of protein-glutamin glutaminase (PGG).

[0050] The present invention also relates to a modified protein-glutamin glutaminase (PGG), produced from the new mutant of Pichia pastoris, and referred as His-Pro-PGG, which has high enzymatic activity and its suitable to be used in the manufacturing of food and food products, such as plant-based beverages, providing stable food products with enhance protein solubility and flavour.

[0051] In the present description, as well as in the claims, the singular forms "a", "an " and "the" include the plural reference unless the context clearly indicates otherwise. The term "about" refers to a deviation of plus / minus 10%, preferably plus / minus 5%. The percentages are expressed in % by weight (wt%), unless stated the contrary. The ranges defined by the terms "between ... and ..." or by the terms “from ...to...” are meant to include also said stated endpoints thereof, and they also include any narrower sub-range.

[0052] The present invention relates to a transformed plasmid or a recombinant plasmid, referred as plasmid pBIZalpha 1.4 HisproPGG, which is characterised because it originates from pBIZalpha and encodes the His pro PGG sequence under AOX1 promoter able to transform the P. pastoris. Said modified plasmid allows transformation of the original P. pastoris and the subsequent obtention of the new Pichia pastoris mutant of the invention, which has the advantage that it does not have antibiotic resistance gene zeocin. The new Pichia pastoris mutant of the invention can be used in enzymatic process, such as in the manufacturing of food, food products, such as plant-based beverage or vegetable milks.

[0053] A plasmid is an isolated nucleic acid construct comprising regulatory sequences operably linked to a coding sequence, suitable for replication and / or expression in a yeast host cell. In the present invention, "plasmid" means a circular or linear extrachromosomal DNA molecule that replicates and transcribes independently of chromosomal DNA.

[0054] Plasmid pBIZalpha 1.4 HisproPGG is a recombinant plasmid.In an embodiment, the plasmid (or construct) referred as plasmid pBIZalpha 1.4 HisproPGG is a modification of the pPICZ, it is suitable as a vector for PGG-expression cassette insertion, and it is characterised because it comprises:

[0055] o a plasmid backbone pBIZa 1.4,

[0056] o a Zeocin antibiotic resistance gene / marker,

[0057] o a A0X1 promoter region,

[0058] o a Ost1-aF region, as an extracellular secretion signal and

[0059] o a target product coding sequence His-Pro-PGG, characterized because it comprises the amino acid sequence SEQ ID NO:1

[0060] In a preferred embodiment, the A0X1 promoter region is inducible upon methanol supplementation,

[0061] The detailed configuration of the plasmid is described in Figure 1 , as a map of the position of all functional elements and other vectors’ components. Likewise, the detailed description of the regions and its position is shown in table 1.

[0062] However, the final construct (plasmid), integrated to the host (p.Pastoris) genome properly assembled is pBIZ1.4 His-Pro-PGG delta Zeo, a recombinant plasmid, and corresponds to the initial plasmid pBIZalpha 1.4 HisproPGG which has been depleted from its selection marker encoded through the antibiotic resistance gene. Thus, in another embodiment, the plasmid (or construct) referred as plasmid pBIZalpha 1.4 HisproPGG is a modification of the pPICZ, and it is characterised because it comprises:

[0063] o a plasmid backbone pBIZa 1.4,

[0064] o a A0X1 promoter region,

[0065] o a Ost1-aF region, as an extracellular secretion signal and

[0066] o a target product coding sequence His-Pro-PGG, characterized because it comprises the amino acid sequence SEQ ID NO:1 and

[0067] o comprises the gene sequence SEQ ID NO:3

[0068] Pichia pastor

[0069] Pichia pastoris is a methylotrophic yeast, currently reclassified as Komagataella pastoris, as disclosed in C. P. Kurtzman, Biotechnological strains of Komagataella (Pichia) pastoris are Komagataella phaffii as determined from multigene sequence analysis, J. Ind. Microbiol. Biotechnol., 2009, 36, 1435-1438.

[0070] Thus, the Pichia pastoris mutant of the invention is characterized in that, the mutant carries a recombinant plasmid expressing protein-glutamine glutaminase, wherein the protein-glutamine glutaminase, is characterised in that it comprises an amino acid encoding sequence SEQ ID NO:1. Furthermore, Pichia pastoris mutant comprises the gene sequence of said proteinglutaminase depleted from its selection marker encoded through the antibiotic resistance gene, which is SEQ ID NO:3. The amino acid encoding sequence SEQ ID NO:1 is characterised because it does not comprise the signal peptide (21 aa), as it is eliminated and it additionally comprises a His tag in N terminal.

[0071] In an embodiment, the yeast cell of the Pichia pastoris mutant of the invention is characterized in that its genome comprises a plasmid (heterologous nucleic acid construct), referred herein as pBIZalpha 1.4 HisproPGG, wherein said plasmid is integrated into the yeast genome and is capable of expressing a gene of interest and the construct is depleted from its selection marker encoded through the antibiotic resistance gene of Zeocin.

[0072] Preferably, the Pichia pastoris mutant of the invention is strain of yeast which genome comprises the DNA sequence of protein-glutamine glutaminase which is SEQ ID NO: 2, a A0X1 region as promoter, wherein the A0X1 region is native promoter for the alcohol oxidase, a pUC o region, Ost-1 -aF region, as an extracellular secretion signal (Ost-1 -aF), wherein the strain comprises an integration of the PGG expression cassette. The sequences were stably integrated. Preferably, the Pichia pastoris mutant of the invention is originate from the strain of yeast PPS9010.

[0073] In a preferred embodiment, the resulted P. pastoris mutant comprised a PPS9010 genome which has a one-copy / gene / location integration of the PGG expression cassette.

[0074] The final plasmid integrated into the host genome (P. pastoris mutant), referred as pBIZ1.4 His-Pro-PGG delta Zeo plasmid is characterized because it encodes the DNA sequence, SEQ ID NO:3.

[0075] In a preferred embodiment, the final construct (plasmid), pBIZ1.4 His-Pro-PGG delta Zeo plasmid, integrated to the host genome of the strain PPS9010, encodes SEQ ID NO:3 and strictly corresponds to the expression cassette depleted from the selection marker encoded through the antibiotic resistance gene of Zeocin.

[0076] The DNA sequence of the expected integration site (native A0X1 locus of the host (p. Pastoris mutant)) is SEQ ID NO:5. Consequently, only one copy number of the expression cassette is expected to arise in the host genome as the system was designed to target the integration of the construct into the corresponding genomic A0X1 locus of the target yeast.

[0077] Protein-glutamine glutaminase (PGG)

[0078] Protein-glutamine glutaminase (PGG) is also known in the art as pro-PG, proteinglutaminase and protein-glutaminase, according to BRENDA database (https: / / www.brenda-enzymes.org / , disclosed in Jeske et al., BRENDA in 2019: a European ELIXIR core data resource, Nucleic Acids Res., 2019, 47, D542-D549).The amino acids sequence of the Chryseobacterium proteolyticum native PGG is SEQ ID NO:4 and was recorded from the reference database UNIPROT (#Q9AQQ8), with Accession number PRJNA471029 (as described in Yamaguchi and Yokoe, 2000),

[0079] PGG belongs to the class EC 3.5.1.44, and it is specific for the hydrolysis of the gammaamide of glutamine substituted at the carboxyl position or both the alpha-amino and carboxyl positions, e.g., L-glutaminylglycine and L-phenylalanyl-L-glutaminylglycine.

[0080] The His-pro-protein-glutamin glutaminase amino acid sequence (305 aa) PGG) is SEQ ID NO: 1. His-Pro-PGG mass: 33378.59 g / mol

[0081] The corresponding DNA sequence for His-Pro-PGG production (PGG expression cassette), which is codon optimized for P. pastoris is SEQ ID NO: 2. When the PGG was prepared from the mutant P. pastoris of the invention, the native signal peptide of the PGG, was supplemented with a signal peptide (a-mating factor) from the plasmid pBIZ1.4, so that the expression of the target enzyme in P. pastoris (K. phaffii) is directed towards an extracellular secretion. A His-tag sequence (consisting in a 6 consecutive histidine motif) was further added in the N-terminal side of the sequence to facilitate detection of the protein product.

[0082] The final construct integrated to the host genome properly assembled is pBIZ1.4 His-Pro-PGG delta Zeo plasmid, which corresponds to the initial plasmid, depleted from its selection marker encoded through the antibiotic resistance gene. The DNA sequence of the final plasmid is SEQ ID NO:3

[0083] The PGG produced from the mutant P. pastoris of the invention is referred as His-Pro-PGG and it is characterised because it comprises the DNA sequence, SEQ ID NO:3.

[0084] The protein-glutamin glutaminase of the invention, His-Pro-PGG, characterised in that it comprises an amino acid sequence SEQ ID NO:1 or homologues thereof having an identity of at least 90%, preferably of at least 93%, more preferably of a least 95%, even more preferably of at least 98%, even more preferably of 100 %.

[0085] "Homologous sequences" or "homologues" means, for the purposes of the invention, that an amino acid sequence has an identity with one of the above amino acid sequences of the monomers of at least 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100%. Preferred are 80% and 90%. Instead of the term "identity", the terms "homolog" or "homology" are used synonymously in the present description. The identity between two nucleic acid sequences or polypeptide sequences is determined by comparison using the program BESTFIT based on the algorithm of Smith, T. F. and Waterman, M. S (Adv. Appl. Math. 2: 482-489 (1981) ) with the following parameters for amino acids: gap creation penalty: 8 and gap extension penalty: 2; and the following parameters for nucleic acids: gap creation penalty: 50 and gap extension penalty: 3. Preferably, the identity between two nucleic acid sequences or polypeptide sequences is defined by the identity of the nucleic acid sequence / polypeptide sequence over the respective entire sequence length as calculated by comparison using the program GAP based on the algorithm of Needleman, S. B.and Wunsch, C. D. Mol. Biol. 48: 443-453) with the following parameters set for amino acids: Gap creation penalty: 8 and Gap extension penalty: 2; and the following parameters for nucleic acids Gap creation penalty: 50 and Gap extension penalty: 3.

[0086] Method to obtain said P. pastoris mutant strain.

[0087] Another aspect of the present invention relates to a method to obtain the P. pastoris mutant of the invention.

[0088] The method to obtain the P. pastoris mutant comprises:

[0089] 1) selecting a wild strain of P. Pastoris,

[0090] 2) transforming the wild strain of P. pastoris with a plasmid, characterised because it is a modified pPICZ plasmid which includes a DNA sequence which is SEQ ID NO: 2, the antibiotic resistance gene for zeocin, a Ost1-aF region, as an extracellular secretion signal and the promoter A0X1 or with the plasmid according to claim 4, to obtain transformed cells of the wild strain of P. pastoris,

[0091] 3) incubating the transformed cells of the P. pastoris strain obtained in step 2, 4) seeding the cells of the transformed P. pastoris strain of step 3 into a plate with the antibiotic zeocin,

[0092] 5) selecting a colony of P. pastoris resistant to the antibiotic zeocin, and

[0093] 6) removing from the P. pastoris cells of step 5) the antibiotic resistance gene for zeocin.

[0094] In a preferred embodiment, the wild strain of P. pastoris is the strain PPS-9010.

[0095] The P. pastoris strain PPS-9010 is commercially available from, for example, the company Atum (USA).

[0096] In an embodiment, the plasmid of step 2 is also referred herein as plasmid pBIZalpha 1.4 HisproPGG, which is characterised because it includes a DNA sequence SEQ ID NO: 2, the antibiotic resistance gene for zeocin, and the promoter A0X1, (as seen in Figure 1), which allows to transform the strain PPS9010. The plasmid pBIZalpha 1.4 HisproPGG, allows the proper insertion of the 4 His-pro-PGG section in the genomic DNA of the P. pastoris strain (preferably strain PPS-9010), providing a “transformed P. pastoris strain”.

[0097] In a preferred embodiment, prior to step 2, the plasmid pBIZalpha 1.4 HisproPGG is previously digested with the restriction enzyme Pmel during a period from 10 up to 32 hours, preferably for at least 16 hours, at temperature from 30 to 45 °C, preferably at a temperature of 37 °C. This step has the advantage that the plasmid is obtained having high purity. Once this digestion step is completed, the plasmid may be further purified with a gel extraction kit, for example, the gel extraction kit available from the company Quimigen.

[0098] In a preferred embodiment, the transformation step 2 is carried out by electroporation.In another embodiment, in the incubation step 3), the cells obtained in step 2) are incubated for a period from 2 to 6 hours, preferably for 3 hours at a temperature from 25-35 °C, preferable at 30 °C, in presence of sorbitol, preferable at a 1M concentration.

[0099] In an embodiment, after incubating the cells in step 3) colonies were obtained and afterwards seeded in step 4) into a plate with the antibiotic zeocin and then, they may be further isolated.

[0100] The colonies obtained in step 4) are generally used to carry out the expression assays. The colonies are selected according to the production of the enzyme His-pro-PGG, defined by SEQ ID NO: 1. The selection test methods are well known to the person skilled in the art, and the SDS-PAGE method may be used in step 5 to select the colonies which express the His-pro-PGG peptide. During step 5) the band corresponding to the enzyme PGG may be confirmed by means of peptide mapping.

[0101] Once step 5) is completed, the best producer clone is identified and selected to perform the removal of the antibiotic gene in step 6).

[0102] In an embodiment, the removal of the antibiotic gene in step 6) comprises, the elimination of the zeocin resistance gen of the cells obtained in step 5 by making competent cells of the best producer clone of the “transformed P. pastoris" with the vector pBGP1 hygro ORE.

[0103] This vector pBGP1 hygro CRE is characterised because encodes the recombinase CRE (Payload), responsible for the recognition of loxP sites in the transformed P. pastoris and the removal of the zeocine resistance gen. vector pBGP1 hygro CRE also comprises a pBGP1 (Backbone), a vector originated from Pichia pastoris which comprises an Autonomous replication sequence (PARS1). The PARS1 allows that the vector is not integrated into the genome but is maintained as an episome (circular and separate from the yeast DNA). This provides and advantage as, it facilitates that vector will be cured (eliminated) once it has done its job.

[0104] Furthermore, vector pBGP1 hygro CRE is also characterised because it comprises a Hygro (Marker): Resistance to Hygromycin B, which allows its entrance in the P.Patoris cells. To perform the selection of the cells receiving the vector pBGP1 hygro CRE, the antibiotic hygromycin is used. Vector pBGP1 hygro CRE beard a selection marker for hygromycin antibiotic. As vector pBGP1 hygro CRE is not inserted in the genome of the mutant P. pastoris, when the P. pastoris mutant strain is then cultured without the antibiotic (hygromycin), the vector is lost in the cell population of the strain when fed without the antibiotic and eliminated from the medium, leading to the mutant P. Pastoris of the invention where the removal of the antibiotic gene has occurred.

[0105] The procedure of step 6) for the removal of the resistance gene, zeocin, may also include additional steps wherein at least one additional culture is carried out in presence and in the absence of the antibiotic hygromycin to provide a suspension culture. Following this at least one supplementation cycle, the cell suspension was seeded in YPD (yeast extract, peptone, dextrose) agar plates and several clones of the mutant P. pastoris were selected for analysis. Afterwards,seeding in YPD agar plates either deprived in the antibiotics, or supplemented in zeocin or supplemented in hygromycin is carried out. A total of 48 clones were eventually selected.

[0106] In a preferred embodiment, the same clone is seeded in YPD agar plates without antibiotic, wherein the seeding is performed in plates, in presence of zeocin and in other plates the seeding is performed in presence of hygromycin.

[0107] The colonies of the P. pastoris that do not grow neither in presence of zeocin nor in the presence of hygromycin, are the colonies which do not include the resistance gene; thus, they are the colonies of the P. pastoris mutant of the invention. Those colonies successfully eliminated both the hygromycin resistance gene and the transient plasmid.

[0108] Expression of the protein by the clones of the mutans of the invention, without the resistance gene may be confirmed by conventional techniques. For example, by means of a 10 mL culture in a BMGY and BMMY medium and inducing with methanol every 24 hours. For this purpose, an SDS PAGE and analysis of activity may be done.

[0109] A BMGY medium comprises: 1% yeast extract, 2% peptone, 0.1M potassium phosphate (pH6.0), 1.34% YNB (Yeast Nitrogen Bases), 0.0004% biotin, and 1% glycerol. It is commercially available through the company GMExpression (Australia).

[0110] A BMMY medium comprises: 1% yeast extract, 2% peptone, 0.1M potassium phosphate (pH6.0), 1.34% YNB (Yeast Nitrogen Bases), 0.0004% biotin, and 0.5% methanol. It is commercially available through the company GMExpression (Australia).

[0111] Activity, such as protein glutaminase enzyme activity, of the obtained P. pastoris mutant samples, may be analysed, for example, after dialysis and digestion of the samples with trypsin following an activity protocol such as that disclosed, for example in, Protein Glutaminase Enzyme Activity Determination Method of CN116200280A or the activity protocol disclosed in in Yamaguchi et al., A novel protein-deamidating enzyme from Chryseobacterium proteolyticum sp. nov., a newly isolated bacterium from soil, Appl. Environ. Microbiol., 2000, 66(8):3337-43. Usually, an enzymatic activity comprised between 4.5 and 5.5 U / mL was obtained. In some embodiments activity values up to 35 U / mL were also obtained. Glutaminase activity is defined as the amount of enzyme capable of hydrolysing the substrate Z-GIn-Gly (benzyloxycarbonyl-glutamine-glycine) under reaction conditions of 37 °C and pH 6.5 for a period of 30 minutes, thereby generating 1 pmol of ammonium ions. This activity corresponds to one unit (U) of glutaminase activity.

[0112] Clones of the strain mutant of the invention may be stored at -80 °C for long time storage, for as long as12 months, preferably for as long as 24 months.

[0113] The P. pastoris mutant strain obtained according to the method of the invention was deposited on May 22, 2024 in the Deutsche Sammlung von Mikroorganismen und Zellkulturen with the number DSM 34997.

[0114] Process for preparing PGG by fermentationAnother aspect of the invention is the use of the Pichia pastoris mutant of the invention for preparing the previously described His-Pro-PGG, which comprises a sequence of 305 amino acids, which is SEQ ID NO: 1.

[0115] A general production process for fermentation of Pichia pastoris is described in the Invitrogen Life Technologies "Pichia Fermentation Guidelines". Main parameters for the fermentation process of Pichia pastoris are:

[0116] • Temperature: 30 °C

[0117] • Dissolved oxygen > 20%

[0118] • pH of 5.0 - 6.0

[0119] • Agitation using an impeller

[0120] • Aeration from 0.1 to 1.0 vvm (Volume of oxygen (L) per volume of fermentation (L) per minute)

[0121] The process for preparing His-Pro-PGG which is SEQ ID N:1 by fermentation of the P. pastoris mutant of the invention comprises the following steps:

[0122] a) culturing the Pichia pastoris mutant in a fermentation medium, preferably the pH of the fermentation media is from 5.0 - 6.0,

[0123] b) adding methanol to the culture obtained in step a), and

[0124] c) removing the cells from the fermentation medium obtained in step b), to obtain a supernatant comprising His-Pro-PGG which is SEQ ID N:1

[0125] In a preferred embodiment the preparation process of His-Pro-PGG which is SEQ ID N:1:

[0126] The fermentation step a) may be carried out at a temperature range from 25 to 35 °C, and / or

[0127] The amount of dissolved oxygen in the fermentation media of step a) is greater than 20% by volume relative to the 100% which is calibrated in the beginning of the fermentation with fresh broth medium and / or

[0128] The fermentation medium may be aerated during the whole fermentation process by means of compressed sterile air and is carried out in a range from 0.1 to 1.0 vvm (Volume of oxygen (L) per volume of fermentation (L) per minute)

[0129] A relative dissolved oxygen level of 100% in water at 25 °C corresponds to 6 mg / L.

[0130] In another preferred embodiment, the fermentation medium of step a) and b) is stirrer, preferably using an impeller, to obtain better homogenisation and better yields.In a further preferred embodiment, antifoam agents may be added in any step of the fermentation process to avoid excess of foam, which may cause denaturation of the secreted protein.

[0131] In a further embodiment, compressed air, synthetic air, or pure oxygen can be used in step b) for gassing. Fermentation typically takes place in the Basal Salts medium. Preferably, the methanol concentration of step b) does not exceed a maximum concentration of 1-2% (v / v), as if this concentration is exceeded, this would be toxic to Pichia pastoris yeast.

[0132] In an embodiment, the fermentation process may be carried out in a bioreactor. In a preferred embodiment a of Pichia pastoris mutant pre-culture may be prepared prior to step a) which can be latter added to inoculate the fermentation media in step a). More preferably a single colony of Pichia pastoris mutant from the agar plate or from a cry stock may be used and deposited in baffled shake flasks to generate the Pichia pastoris mutant pre-culture.

[0133] Afterwards, the aforementioned Pichia pastoris mutant pre-culture is added to the fermentation medium, then it may be grown during step b) in a BMGY ora BMN medium. Pichia pastoris mutant cell growth may be monitored at various time points by using a visible light spectrophotometer wherein the absorbance is set at 600 nm (ODeoo).

[0134] The BMN medium of step b) is used in a Buffered Minimal Methanol Medium (BMM) as disclosed, for example, in Cold Spring Harbor Protocols, 2021, doi:10.1101 / pdb.rec105247.

[0135] The preparation of the Pichia pastoris mutant pre-culture fermentation, referred as preculture fermentation step, is performed at a temperature range from, 25-35 °C, preferably at 30 °C, at a stirring speed from 250 to 300 rpm, for at least 10 h, preferably from 16 to 24 h, preferably until an ODeoo of from 2 to 6 is obtain. Thus, in a preferred embodiment, in step a) the bioreactor may be inoculated with a range from 5 - 10% v / v of the Pichia pastoris mutant pre-culture fermentation product. The batch typically lasts for 18 - 24 h.

[0136] When fermentation is completed in step b), the full culture volume may be recovered and centrifuged to eliminate the undesirable biomass. Preferably, afterwards in step c) the obtained product maybe microfiltered to eliminate any remining cell in the recovered liquid, so the full culture volume may be recovered as a supernatant, for example by centrifugation means. The supernatant may be stored at temperatures from -40 to -15 °C.

[0137] The mutant P. pastoris strain of the invention has the advantage that it exhibits increased enzyme activity as compared to the parent P. pastoris strain.

[0138] The results obtained with the aforementioned fermentation process showed that the His-Pro-PGG enzyme activity of the fermentation supernatant obtained from the Pichia pastoris mutant of the invention, reached at least 16 U / rnL, and values as high as 35 U / rnL which is a surprising result compared to prior art strains of P. pastoris, as for example, the strain disclosed in CN-A-116200280 which produced an enzyme activity of 6.0 U / rnL.

[0139] The His-pro-PGG enzyme of the invention is a precursor of PGG and has the advantage that is more stable than PGG (the enzymatic activity is maintained over time), thus it is easier tohandle and to store for industrial applications. The His-pro-PGG enzyme and can be obtained as a powder by means of a freeze-dry or spray drying process. The His-pro-PGG enzyme powder has the further advantage that is suitable for food grade use without the presence of any preservative.

[0140] Thus, in a preferred embodiment of the invention, His-pro-PGG enzyme is suitable as precursor or intermediate of PGG and / or as an industrial alternative to PGG due to its improved stability and industrial handling.

[0141] The mature PGG enzyme has the disadvantage that, when used — particularly at the industrial scale — it is employed in liquid form. This liquid form may lose more than 50% of its enzymatic activity at the aforementioned temperatures. Advantagously, the His-pro-PGG enzyme it can be stored as a dried powder at a temperature range from -10 to 25 °C, preferably from 0-20 °C, while maintaining its stability as well as its enzymatic activity. In a preferred embodiment, the enzymatic activity of the His-pro-PGG enzyme, preferably as a powder, is maintained up to 99% after 1 month of storage at the aforementioned temperatures. Preferably, the enzymatic activity is maintained up to 95% after 6 months of storage at the aforementioned temperatures.

[0142] The His-pro-PGG enzyme is later converted to a mature active enzyme by treatment with a protease. Typically, 1 II (according to S-AZCAS 06 / 20 method from Mega zymes) of bacterial metalloprotease per litre of raw liquid enzyme it is added, and the mixture is usually incubated for about 2 hours at a temperature of about 37 °C.

[0143] Uses of the PGG as obtained from the mutant strain of P.pastoris of the invention.

[0144] The enzyme PGG is used in food manufacturing.

[0145] The main function of this protein is the deamidation of glutaminyl residues in proteins. The effects of deamidation on proteins result in protein unfolding exposing previously buried hydrophobic sites and reducing intra / intermolecular hydrogen bonding, rebalancing the hydrophilic-hydrophobic characteristics of food proteins, impacting their ability to emulsify and foam by increasing the concentration of soluble proteins, which aids in creating a stable layer at interfaces between oil and water or air and water, and altering protein structures and conformations, which can potentially change the flavour of food proteins.

[0146] In an embodiment, the His-Pro-PGG enzyme obtained from the P.pastoris mutant strain of the invention or from the P.pastoris mutant strain obtained from method of the invention is used to improve protein solubility. Solubility is influenced by factors such as amino acid composition and sequence. However, certain proteins, particularly those rich in nonpolar amino acids and glutamine residues, tend to aggregate, decreasing solubility. Specifically, deamidation targets glutamine residues, forming short peptide sidechains and converting amide groups into negatively charged carboxyl groups. This alteration in charge enhances the hydrophilicity of the proteinsurface and lowers the isoelectric point, ultimately enhancing solubility. In a preferred embodiment His-Pro-PGG enzyme improves the solubility of proteins in protein-containing flours, selected from the group consisting of wheat gluten, wheat protein, oat protein, rice protein, almond protein, walnut protein, pea protein, quinoa protein and soy protein isolate.

[0147] In an embodiment, the His-Pro-PGG enzyme obtained from the P.pastoris mutant strain of the invention or from the P.pastoris mutant strain obtained from method of the invention is used to improve emulsification and foaming of proteins in beverages, preferably in vegetable beverages. Additionally, converting glutamine residues into glutamic acid introduces negative charges to the protein, generating repulsive forces at these interfaces. This helps to slow down droplet movement, leading to improved stabilization. Thus, His-Pro-PGG enzyme is suitable to improve the emulsification of proteins, selected from the group consisting of, milk proteins, wheat gluten, a-zein, oat protein, soja protein, almond protein and coconut protein. In a preferred embodiment, the His-Pro-PGG enzyme is suitable to improve the water holding capacity or oil holding capacity or foaming capacity of coconut protein, wheat gluten protein, wheat protein, oat protein, rice protein, pea protein, almond protein, rice protein, walnut protein, quinoa protein and soy protein isolate.

[0148] In an embodiment, the His-Pro-PGG enzyme obtained from the P. pastoris mutant strain of the invention or from the P. pastoris mutant strain obtained from method of the invention is used to enhance flavour. Ammonia released from food proteins, along with their affinity for flavour compounds, contributes to the creation of various volatile components. Studies have shown that deamidation, reduces the protein's ability to bind to flavours like vanillin and maltol, while improving solubility under acidic conditions. This process also diminishes flavour deterioration in high-protein water-based foods, such as coconut proteins. Furthermore, deamidation enhances the umami taste and reduces bitterness in protein hydrolysates. However, the binding strength of flavours with deamidated proteins differs from that of unmodified proteins, possibly due to changes in binding mechanisms. Analytical methods alone cannot fully predict consumer perception of flavour-protein interactions, necessitating further research using sensory evaluation techniques. In a preferred embodiment, the enzyme PGG obtained from the P.pastoris mutant strain of the invention or from the P.pastoris mutant strain obtained from method of the invention is suitable to enhance flavours, of cow’s milk, dairy products or plant-based drinks o plant milks, selected from the list consisting of soymilk, soy protein isolate, oat milk, rice milk, almond milk hazelnut milk and coconut milk.

[0149] In an embodiment, the enzyme PGG obtained from the P.pastoris mutant strain and / or from the method of the invention is suitable for reducing the allergenic potential of specific proteins, such as for the manufacturing of low-allergenic gluten products, gluten free product (foods that contain less than 20 parts per million (ppm) of gluten), a protein found in wheat, barley, rye, and their hybrids.The present invention may be defined according to the following embodiments:

[0150] 1. A Pichia pastoris mutant yeast strain characterized in that, the mutant carries a recombinant plasmid expressing protein-glutamine glutaminase, wherein the protein-glutamine glutaminase, is characterised in that it comprises an amino acid encoding sequence SEQ ID NO:1., and preferably the gene sequence of said protein glutaminase is SEQ ID NO:3 and / or wherein the strain of the mutant P. pastoris the PPS9010 strain.

[0151] 2. The Pichia pastoris mutant yeast strain according to embodiment 1, wherein the plasmid is a modification of the plasmid pPICZ, and it is characterised because it comprises:

[0152] o a plasmid backbone pBIZa 1.4,

[0153] o an A0X1 promoter region,

[0154] o a Ost1-aF region, as an extracellular secretion signal and

[0155] o a target product coding sequence protein-glutamine glutaminase, characterized because it comprises the amino acid sequence SEQ ID NO:1, and

[0156] wherein the recombinant plasmid has been depleted from its selection marker encoded through the antibiotic resistance gene zeocin.

[0157] 3. The Pichia pastoris mutant strain according to anyone of embodiment 1-2, deposited by the company CYGYC Biocon S.L. on May 22, 2024, in the Deutsche Sammlung von Mikroorganismen und Zellkulturen with the number DSM 34997.

[0158] 4. A recombinant plasmid, wherein the plasmid is a modification of the plasmid pPICZ, and it is characterised because it comprises:

[0159] a plasmid backbone pBIZa 1.4,

[0160] an AOX1 promoter region,

[0161] a Zeocin antibiotic resistance gene,

[0162] a Ost1-aF region, as an extracellular secretion signal and

[0163] a target product coding sequence protein-glutamine glutaminase, characterized because it comprises the amino acid sequence SEQ ID NO:1.

[0164] 5. A method to obtain the P. pastoris mutant strain yeast of embodiments 1-3 comprising:

[0165] 1. selecting a wild strain of P. Pastoris,

[0166] 2. transforming the wild strain of P. pastoris with a recombinant plasmid, characterised because it is a modified pPICZ plasmid which includes a DNA sequence which is SEQ ID NO: 2, the antibiotic resistance gene for zeocin, a Ost1-aF region, as an extracellular secretion signal and the promoter A0X1 or with the plasmid according to embodiment 4, to obtain transformed cells of the wild strain of P. pastoris,3. incubating the cells of the transformed P. pastoris strain obtained in step 2, 4. seeding the cells of the transformed P. pastoris strain of step 3 into a plate with the antibiotic zeocin,

[0167] 5. selecting a colony of P. pastoris resistant to the antibiotic zeocin, and

[0168] 6. removing from the P. pastoris cells of step 5) the antibiotic resistance gene for zeocin.

[0169] 6. The method according to embodiment 5, wherein the wild strain of P. pastoris is the strain PPS-9010.

[0170] 7. The method according to any one of embodiments 5 to 6, wherein the plasmid of step 2) prior to its use in step 2), is previously digested with the restriction enzyme Pmel for at least 10 hours, preferably from 12 to 24 hours and / or at a temperature range from 30-43 °C.

[0171] 8. The method according to any one of embodiments 5-7, wherein the plasmid of step 2) is purified with a gel extraction kit after the digestion.

[0172] 9. The method according to any one of embodiments 5 to 8, wherein the transformation of step 2) is carried out by electroporation.

[0173] 10. Use of the Pichia pastoris mutant according to any of the embodiments 1-3 or the Pichia pastoris mutant obtained from the method according to anyone of embodiments 5-9, for preparing protein-glutamine glutaminase (PGG) and / or in the manufacturing of food.

[0174] 11. Use of the Pichia pastoris mutant according to any of the embodiments 1-3 or the Pichia pastoris mutant obtained from the method according to anyone of embodiments 5-9, in a method for the production food or food products selected from the list consisting of, of plant-based analogues of milk, milk products, plant-based beverages, low-allergenic gluten products, gluten free products and vegetable drinks.

[0175] 12. A protein-glutamine glutaminase characterised in that said protein comprises an amino acid sequence SEQ ID NO:1 or homologues thereof having an identity of at least 90%.

[0176] 13. The protein-glutamine glutaminase according to embodiment 12, characterised because it comprises a histidine tag in the N terminal position and the absence of the signal peptide (21 aa).

[0177] 14. The protein-glutamine glutaminase according to any one of the claims 12-13 for use as glutaminase.15. A recombinant plasmid which comprises a DNA sequence, SEQ ID NO:3

[0178] 16. A Process to obtain the protein-glutamine glutaminase according to anyone of embodiments 12-14, characterized in that, the process comprises the use the Pichia pastoris mutant strain according to anyone of embodiments 1-3, or the Pichia pastoris mutant obtained from the method according to anyone of embodiments 5-9, as a fermentation strain.

[0179] 17. The process to obtain protein-glutamine glutaminase according to embodiments 16 which comprises cultivating the Pichia pastoris mutant in a culture medium to produce glutaminase in the culture.

[0180] 18. The process to protein-glutamine glutaminase according to anyone of embodiments 16-17, which comprises the following steps:

[0181] a) culturing the fermentation strain Pichia pastoris mutant in a fermentation medium, b) adding methanol to the culture obtained in step a), and

[0182] a) removing the cells from the fermentation medium obtained in step b), to obtain a supernatant comprising the targeted protein-glutamine glutaminase, and b) optionally providing the protein-glutamine glutaminase obtained of step c) as a powder by freeze drying or spray drying processes.

[0183] 19. Use of the protein-glutamine glutaminase according to any one of the embodiments 12-15 or to the protein-glutamine glutaminase obtained according to any one of the embodiments 16-18, as food enzyme, preferably in the manufacturing of food products, plant-based beverages and vegetable drinks.

[0184] 20. Use of the protein-glutamine glutaminase according to any one of the embodiments 12-15 or to the protein-glutamine glutaminase obtained according to any one of the embodiments 16-18, in a process for deaminating vegetable proteins, preferably to improve their proteins stability and or solubility in plant-based beverages.

[0185] 21. A DNA sequence which encodes a protein according to any one of the embodiments 12-15, or to the protein-glutamine glutaminase obtained according to any one of the embodiments 16-18, which comprises the nucleotide sequence according to SEQ ID N:3.

[0186] 22. The Pichia pastoris mutant yeast strain, strain characterized in that, the mutant carries a recombinant plasmid expressing protein-glutamine glutaminase, wherein the plasmid is a modification of the plasmid pPICZ, and it is characterised because it comprises:

[0187] a plasmid backbone pBIZa 1.4,an AOX1 promoter region,

[0188] a Zeocin antibiotic resistance gene,

[0189] a Ost1-aF region, as an extracellular secretion signal and

[0190] containing a DNA sequence SEQ NO:2

[0191] EXAMPLES

[0192] The techniques and DNA recombinant methods applied herein, are described in detail in Sambrook and Russell, Molecular cloning 3rd Ed. Cold Spring Harbor Laboratory Press, Cold spring Harbor, New York, 2001.

[0193] BMGY medium comprises: 1% yeast extract, 2% peptone, 0.1M potassium phosphate (pH6.0), 1.34% YNB (Yeast Nitrogen Bases), 0.0004% biotin, and 1% glycerol. It is commercially available through the company GMExpression (Australia).

[0194] Example 1: Obtention of the mutant strain

[0195] A. Selection of a wild strain of P. pastoris

[0196] Wild strain PPS-9010 (Atum) was chosen as starting P. pastoris strain.

[0197] B. Transformation

[0198] Plasmid pBIZalpha 1.4 HisproPGG was used in a process to transform the strain PPS9010 into a transformed P. pastoris strain. Prior to the transformation step, and to allow the proper insertion of the plasmid to the genomic DNA of the P. pastoris strain PPS9010. Plasmid pBIZalpha 1.4 HisproPGG was digested with the restriction enzyme Pmel (5 U / pl) for 16 hours at 37 °C and later purified with a gel extraction kit (Quimigen).

[0199] Afterwards, an aliquot of electro competent cells of P. pastoris strain PPS9010 was mixed with approximately 2 pg of purified DNA into an electroporation cuvette and was electroporated into the Extender Plus Genepulser II System (2 mm, 2 kV, 25 pF, 200 Q).

[0200] Subsequently, the cells obtained were then incubated for 3 hours at 30 °C in presence of sorbitol 1M and seeded into a plate with the antibiotic zeocin. Isolated colonies of the cells were used for the master plate and to carry out the expression assays.

[0201] B.1.- PGG sequence: design, synthesis, and cloning

[0202] The pro-protein glutaminase amino acid sequence of 305 amino acids is SEQ ID NO: 1. The DNA sequence (SEQ ID NO: 2) was codon-optimized in the plasmid pPICZ and the plasmid of the invention for P. pastoris and synthetized by Genscript.

[0203] The predicted molecular weight for the His-Pro-PGG is 33.5 kDa and it has an Isoelectric point of 8.4. The sequence of the mature (activated) PGG SEQ ID, N:4. On the sequence of themature PGG, there is one potential / V-glycosylation site at position 123 and 5 potential O-glycosylation sites in positions 22, 26, 29, 30, and 173.

[0204] The methanol-induced promoter A0X1 was used.

[0205] B.2.- Genetic Construct pBIZa 1.4 HisproPGG (plasmid)

[0206] The plasmid pBIZalpha 1.4 HisproPGG, which comprised the insert HisproPGG Chryseobacterium proteolyticum, the antibiotic resistance gene for Zeocin, and the promoter A0X1 was used.

[0207] In Figure 1 it is shown the plasmid of the invention map, plasmid pBIZalpha 1.4 HisproPGG map, which encodes the His proPGG sequence under A0X1 promoter, which was used to transform a wild strain of P. pastoris, preferably strain PPS-9010

[0208] B.3.- Transformation by electroporation

[0209] A method for electroporation was used, for example, in Lin-Cereghino et al., Competent Cell Preparation and Transformation of Pichia pastoris. Methods Mol. Biol., 2022, 2513, 113-120 An aliquot of electro competent cells of Pichia pastoris was mixed with approximately 2 pg of purified DNA of the plasmid into an electroporation cuvette and was electroporated into the Extender Plus Genepulser II System (2 mm, 2 kV, 25 pF, 200 Q).

[0210] Cells of transformed P. pastoris strain PPS-9010 were then incubated for 3 hours at 30 °C in presence of sorbitol 1M and seeded into a plate with the antibiotic zeocin. Isolated colonies of the above cells were used for the master plate and to carry out the expression assays.

[0211] B.4.- Clone selection of pBIZalpha 1.4 HisproPGG clones

[0212] Several colonies appeared after the transformation procedure. 10 clones were selected to continue with the expression assays. Cultures were fed every 24 hours with 1% methanol and after 72 hours they were centrifuged. All clones arrived at optical densities between 40 and 50. Final supernatants were then analysed by SDS PAGE.

[0213] To check if productivity could be increased by inducing more times the cultures, new cultures were done with these clones. This time induction was run twice per day. After 72 hours of culture, optical densities were between 42 and 60 in all clones. SDS PAGE showed an increase in the intensity of the band corresponding to the size of the HisproPGG protein. In was confirmed that the band corresponded to the protein HisproPGG by peptide mapping.

[0214] C.- Removal of the antibiotic resistance gene

[0215] The best producer clone was selected to make the removal of the antibiotic gene. The first step was to make competent cells of the previously developed strain PPS9010 pBIZ1.4 HisproPGG (prior to the elimination of the antibiotic resistance) best producer clone, by means of transformation process using the vector pBGP1 Hygro CRE (Figure 2). This vector encoded forthe recombinase CRE, responsible for the recognition of loxP sites and the removal of the sequences in between. To make the selection of the cells containing this vector, the antibiotic hygromycin was used. As this vector was not inserted in the genome of P. pastoris, when the strain was cultured without the antibiotic, plasmid was lost in the cell population of the strain when fed without the antibiotic and eliminated from the medium.

[0216] The procedure for the removal of the resistance gene includes a series of cultures in presence of the antibiotic hygromycin and without. At the end, the suspension was seeded in YPD agar plates, and several clones were selected to be analysed.

[0217] The same clone was seeded in YPD agar plates without antibiotic, in plates in presence of zeocin and in plates in presence of hygromycin. A total of 48 clones were picked.

[0218] Along with the 48 picked clones, only 6 were able to grow in presence of Zeocin, and only 1 still contained the episomal vector pBGP1 Hygro CRE. Those colonies that did not grow neither in presence of zeocin nor hygromycin, eliminated the resistance gene successfully.

[0219] Expression of the protein by the clones without the resistance gene was confirmed with a 10 mL culture in medium BMGY and BMMY and inducing with methanol every 24 hours. For this purpose, an SDS PAGE and analysis of activity were done.

[0220] Activity of the samples was analysed after dialysis and digestion with trypsin following the protocol of activity (Yamaguchi et al., op. cit.) or protocol of CN116200280A. The result of the activity obtained was comprised between 5 and 35 U / rnL.

[0221] As the removal of the resistance gene and the expression of the protein was confirmed, Master Cell Bank with three clones was done and stored at -80 °C for long time storage.

[0222] The P. pastoris mutant strain obtained according to this method was deposited by the company CYGYC Biocon S.L. on May 22, 2024, in the Deutsche Sammlung von Mikroorganismen und Zellkulturen with the number DSM 34997

[0223] Example 2: Obtention of PGG by fermentation

[0224] 100 pl of the cryostock of Pichia pastoris mutant obtained in Example 1 was inoculated in an Erlenmeyer containing 400 mL of BMGY medium, and it was incubated at 30 °C, 250 rpm for 1 day.

[0225] The following day the culture was used to inoculate a 5 L bioreactor containing 3 L of BMM medium, and was incubated at 30 °C, 250rpm. 1% methanol was added every 5 hours. After 48 hours of induced expression, the cells were removed by centrifugation to obtain a fermentation supernatant; the fermentation supernatant was tested for protein-glutamine glutaminase activity.

[0226] In a preferred embodiment, the His-Pro-protein-glutamine glutaminase obtained in the supernatant, may be obtained as a powder by freeze drying or spray drying processes, as its stability and handling storage is improved, particularly when used at industrial scale.

[0227] No evidence of toxicity was observed.Additionally , according to the results obtained from the alignments and taking into account the most recent scientific recommendations on the interpretation of such data, it was concluded that the PGG enzyme of the invention shows no allergy concern and it is safe for use in food processing, with no indication of adverse effects or reactions.

[0228] The results showed that the PGG enzyme activity of fermentation supernatant obtained from the Pichia pastoris mutant of the invention reached 35 U / rnL, activity determined according to Yamaguchi et al., op. cit. or CN116200280A.

[0229] Example 3: preparation process of an oat-based beverage with the his-pro-ppg enzyme of the invention.

[0230] In the first step of the production of plant-based analogues of milk and milk products or plant-based beverages, the His-Pro-PGG enzyme is converted (activated) into the mature or activated PGG enzyme by hydrolysis in the presence of a protease. Preferably, the protease uses is a metallo endopeptidasa (3.4.24), serine endopeptidase (3.4.21) or mixtures thereof Once the mature or activated PGG is obtained from the His-Pro-PGG enzyme, the activated PGG is added at a second step, the stabilization step, thus it can perform its technological function during the initial incubation of the plant-based raw materials. PGG has the advantage that it does not exert any (unintentional) enzymatic activity in the final foods.

[0231] So far, the first and second step are carried out separately, as if performed in-situ the result of the process is worse and, in some occasions, the amount of the resulting product is very low.

[0232] Preparation of the oat beverage

[0233] A 15% oat beverage was produced from an oat flour containing 17% protein, wherein the protein was enzymatic incubated using activated PGG, previously obtained from His-Pro-PGG enzyme of the invention.

[0234] Water was first preheated to 60 °C and CaCI2was added at a final concentration of 50 ppm. An alpha-amylase was then added at 0.25% (d.w.m., based on flour), followed by the addition of oat flour (previously incubated with the His-Pro-PGG enzyme of the invention) to reach a final concentration of 15%. The slurry was incubated at 60 °C for 5 minutes and subsequently heated to 80 °C at a rate of 2 °C / min, where it was maintained for 30 minutes to promote starch liquefaction. The mixture was then cooled to 60 °C, and glucoamylase was added at 0.1% (d.w.m., flour), together with protein glutaminase at the specified dosage. The mixture was incubated at 60 °C for 60 minutes, after which the enzymes were inactivated at 90 °C for 5 minutes. Finally, the oat beverage was filtered and its pH adjusted to 7.2 using calcium carbonateThe protein solubility and stability of the oat-based beverage was assessed when the oat beverage was mixed with coffee at a pH of 5.4, as it is a disadvantage behaviour observed with oat-based beverage and with another plant-based beverage.

[0235] Equal volumes of the beverage and coffee by mixing. Specifically, 50 mL of the oat beverage were combined with 50 mL of coffee and the pH adjusted to a pH of 5.4. The resulting mixtures are described in table 1 and were observed to assess (see criterium in table 2) beverage stability under acidic coffee conditions, as shown in table 1.

[0236]

[0237] Table 1

[0238]

[0239] Table 2

[0240] Thus, it can be concluded that samples without His-Pro-PGG showed clear phase separation and the formation of large clogs or aggregates, indicating poor protein solubility under acidic conditions.The addition of at least 7 II His-Pro-PGG / L of oat beverage allows the samples to partially stabilize when mixed with coffee at pH 5.4; however, they are not fully homogeneous and still present small clogs or fine aggregates, suggesting limited stability.

[0241] Increasing the dosage of protein glutaminase to 30 II PGG / L prevents beverage separation when mixed with acidic coffee at pH 5.4. Under these conditions, the samples remain uniform after mixing, with no visible clogs or sediment, indicating good protein solubility and high stability in coffee.

[0242] Thus, the protein solubility is increased under acidic conditions as well as the stability of the oat beverage even at low pH values, when the oat beverage was produced with the His-Pro-PGG of the invention.SEQUENCES

[0243] SEQ ID NO: 1

[0244] HHHHHHDSNGNQEINGKEKLSVNDSKLKDFGKTVPVGIDEENGMIKVSFMLTAQFYEIKPTKEN EQYIGMLRQAVKNESPVHIFLKPNSNEIGKVESASPEDVRYFKTILTKEVKGQTNKLASVIPDVA TLNSLFNQIKNQSCGTSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQFVYGNLKAS TGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCGSASVSSYA NTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0245] SEQ ID NO: 2

[0246] CATCATCATCATCATCATGATTCTAACGGTAACCAAGAAATTAATGGTAAAGAGAAGTTGTC TGTTAACGATTCTAAGTTGAAGGATTTCGGTAAAACTGTTCCTGTTGGTATCGATGAAGAGA ACGGTATGATCAAAGTTTCTTTTATGTTGACTGCTCAATTCTACGAGATTAAGCCAACTAAG GAAAACGAGCAATACATCGGAATGTTGAGACAAGCTGTTAAGAACGAATCTCCTGTTCATA TTTTCTTGAAGCCAAACTCTAACGAAATTGGTAAAGTTGAGTCTGCTTCTCCTGAAGATGTT AGATACTTCAAGACTATCTTGACTAAGGAGGTTAAAGGTCAAACTAACAAATTGGCTTCTGT TATTCCAGATGTTGCTACTTTGAACTCTTTGTTTAATCAAATTAAGAACCAATCTTGTGGTAC TTCTACTGCTTCTTCTCCTTGTATTACTTTCAGATACCCAGTTGATGGTTGTTATGCTAGAGC CCATAAGATGAGACAAATTTTGATGAACAACGGTTACGATTGTGAGAAGCAATTCGTTTACG GTAATTTGAAGGCTTCTACTGGTACTTGTTGTGTTGCTTGGTCTTACCACGTTGCTATTTTG GTTTCTTATAAGAACGCTTCTGGTGTTACTGAAAAGAGAATCATCGATCCTTCTTTGTTCTCT TCTGGTCCAGTTACTGATACTGCTTGGAGAAATGCTTGTGTTAACACTTCTTGTGGTTCTGC TTCTGTTTCTTCTTATGCTAATACTGCTGGTAACGTTTACTATAGATCCCCATCTAACTCTTA CTTGTACGATAACAATTTGATTAACACTAATTGTGTTTTGACTAAATTTTCTTTGTTGTCTGG TTGTTCTCCATCTCCTGCTCCAGATGTTTCTTCTTGTGGTTTCTAA

[0247] SEQ ID NO: 3

[0248] TGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTC CATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTG GCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGC TGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATC GTCTTGAGTGCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACT ACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTT GTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGACATGTCGCCGGCGCTAGAGATCTAACATCCAAAGACGAAAGGTTGAATGAAA CCTTTTTGCCATCCGACATCCACAGGTCCATTCTCACACATAAGTGCCAAACGCAACAGGA GGGGATACACTAGCAGCAGACCGTTGCAAACGCAGGACCTCCACTCCTCTTCTCCTCAAC ACCCACTTTTGCCATCGAAAAACCAGCCCAGTTATTGGGCTTGATTGGAGCTCGCTCATTC CAATTCCTTCTATTAGGCTACTAACACCATGACTTTATTAGCCTGTCTATCCTGGCCCCCCT GGCGAGGTTCATGTTTGTTTATTTCCGAATGCAACAAGCTCCGCATTACACCCGAACATCA CTCCAGATGAGGGCTTTCTGAGTGTGGGGTCAAATAGTTTCATGTTCCCCAAATGGCCCAA AACTGACAGTTTAAACGCTGTCTTGGAACCTAATATGACAAAAGCGTGATCTCATCCAAGAT GAACTAAGTTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAG TCGGCATACCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAAT GCTTAGCGCAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATG GGGAAACACCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCT GGTGGGAATACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACT TGACAGCAATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTA TTAGCTTACTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTA ACGACAACTTGAGAAGATCAAAAAACAACTAATTATTCGGCGCGCCGAAACGATGAGGCAG GTTTGGTTCTCTTGGATTGTGGGATTGTTCCTATGTTTTTTCAACGTGTCTTCTGCTGCTCC AGTCAACACTACAACAGAAGATGAAACGGCACAAATTCCGGCTGAAGCTGTCATCGGTTAC TCAGATTTAGAAGGGGATTTCGATGTTGCTGTTTTGCCATTTTCCAACAGCACAAATAACGG GTTATTGTTTATAAATACTACTATTGCCAGCATTGCTGCTAAAGAAGAAGGGGTATCTCTCG AGAAAAGACATCATCATCATCATCATGATTCTAACGGTAACCAAGAAATTAATGGTAAAGAG AAGTTGTCTGTTAACGATTCTAAGTTGAAGGATTTCGGTAAAACTGTTCCTGTTGGTATCGA TGAAGAGAACGGTATGATCAAAGTTTCTTTTATGTTGACTGCTCAATTCTACGAGATTAAGC CAACTAAGGAAAACGAGCAATACATCGGAATGTTGAGACAAGCTGTTAAGAACGAATCTCC TGTTCATATTTTCTTGAAGCCAAACTCTAACGAAATTGGTAAAGTTGAGTCTGCTTCTCCTG AAGATGTTAGATACTTCAAGACTATCTTGACTAAGGAGGTTAAAGGTCAAACTAACAAATTG GCTTCTGTTATTCCAGATGTTGCTACTTTGAACTCTTTGTTTAATCAAATTAAGAACCAATCT TGTGGTACTTCTACTGCTTCTTCTCCTTGTATTACTTTCAGATACCCAGTTGATGGTTGTTAT GCTAGAGCCCATAAGATGAGACAAATTTTGATGAACAACGGTTACGATTGTGAGAAGCAAT TCGTTTACGGTAATTTGAAGGCTTCTACTGGTACTTGTTGTGTTGCTTGGTCTTACCACGTT GCTATTTTGGTTTCTTATAAGAACGCTTCTGGTGTTACTGAAAAGAGAATCATCGATCCTTC TTTGTTCTCTTCTGGTCCAGTTACTGATACTGCTTGGAGAAATGCTTGTGTTAACACTTCTT GTGGTTCTGCTTCTGTTTCTTCTTATGCTAATACTGCTGGTAACGTTTACTATAGATCCCCAT CTAACTCTTACTTGTACGATAACAATTTGATTAACACTAATTGTGTTTTGACTAAATTTTCTTTGTTGTCTGGTTGTTCTCCATCTCCTGCTCCAGATGTTTCTTCTTGTGGTTTCTAACATTAATC AAGAGGATGTCAGAATGCCATTTGCCTGAGAGATGCAGGCTTCATTTTTGATACTTTTTTAT TTGTAACCTATATAGTATAGGATTTTTTTTGTCATTTTGTTTCTTCTCGTACGAGCTTGCTCC TGATCAGCCTATCTCGCAGCTGATGAATATCTTGTGGTAGGGGTTTGGGAAAATCATTCGA GTTTGATGTTTTTCTTGGTATTTCCCACTCCTCTTCAGAGTACAGAAGATTAAGTGACACGT TCGTTTGTGCAAGCTTCAACGATGCCAAAAGGGTATAATAAGCGTCATTTGCAGCATTGTG AAGAAAACTATGTGGCAAGCCAAGCCTGCGAAGAATGTACCATGGATAACTTCGTATAGCA TACATTATACGAAGTTATGCG

[0249] SEQ ID N:4

[0250] LASVIPDVATLNSLFNQIKNQSCGTSTASSPCITFRYPVDGCYARAHKMRQILMNNGYDCEKQF VYGNLKASTGTCCVAWSYHVAILVSYKNASGVTEKRIIDPSLFSSGPVTDTAWRNACVNTSCG SASVSSYANTAGNVYYRSPSNSYLYDNNLINTNCVLTKFSLLSGCSPSPAPDVSSCGF

[0251] SEQ ID N:5

[0252] TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTTCCAAAAGTCGGCATA CCGTTTGTCTTGTTTGGTATTGATTGACGAATGCTCAAAAATAATCTCATTAATGCTTAGCG CAGTCTCTCTATCGCTTCTGAACCCCGGTGCACCTGTGCCGAAACGCAAATGGGGAAACA CCCGCTTTTTGGATGATTATGCATTGTCTCCACATTGTATGCTTCCAAGATTCTGGTGGGAA TACTGCTGATAGCCTAACGTTCATGATCAAAATTTAACTGTTCTAACCCCTACTTGACAGCA ATATATAAACAGAAGGAAGCTGCCCTGTCTTAAACCTTTTTTTTTATCATCATTATTAGCTTA CTTTCATAATTGCGACTGGTTCCAATTGACAAGCTTTTGATTTTAACGACTTTTAACGACAAC TTGAGAAGATCAAAAAACAACTAATTATTCGAAACG

Claims

1. A Pichia pastoris mutant yeast strain characterized in that, the mutant carries a recombinant plasmid expressing protein-glutamine glutaminase, wherein the protein-glutamine glutaminase, is characterised in that it comprises an amino acid encoding sequence SEQ ID NO:1.

2. The Pichia pastoris mutant yeast strain according to claim 1, wherein the recombinant plasmid is a modification of the plasmid pPICZ, and it is characterised because it comprises:a plasmid backbone pBIZa 1.4,an AOX1 promoter region,a Ost1-aF region, as an extracellular secretion signal anda target product coding sequence protein-glutamine glutaminase, characterized because it comprises the amino acid sequence SEQ ID NO:1, andwherein the recombinant plasmid has been depleted from its selection marker encoded through the antibiotic resistance gene zeocin.

3. The Pichia pastoris mutant strain according to anyone of claims 1-2, deposited by the company CYGYC Biocon S.L. on May 22, 2024, in the Deutsche Sammlung von Mikroorganismen und Zellkulturen with the number DSM 34997.

4. A recombinant plasmid, wherein the plasmid is a modification of the plasmid pPICZ, and it is characterised because it comprises:a plasmid backbone pBIZa 1.4,an AOX1 promoter region,a Zeocin antibiotic resistance gene,a Ost1-aF region, as an extracellular secretion signal anda target product coding sequence protein-glutamine glutaminase, characterized because it comprises the amino acid sequence SEQ ID NO:1.

5. A method to obtain the P. pastoris mutant strain yeast of claims 1-3 comprising:1) selecting a wild strain of P. Pastoris,2) transforming the wild strain of P. pastoris with a recombinant plasmid, characterised because it is a modified pPICZ plasmid which includes a DNA sequence which is SEQ ID NO: 2, the antibiotic resistance gene for zeocin, a Ost1-aF region, as an extracellular secretion signal and the promoter A0X1 or with the plasmid according to claim 4, to obtain transformed cells of the wild strain of P. pastoris, 3) incubating the cells of the transformed P. pastoris strain obtained in step 2,4) seeding the cells of the transformed P. pastoris strain of step 3) into a plate with the antibiotic zeocin,5) selecting a colony of P. pastoris resistant to the antibiotic zeocin, and6) removing from the P. pastoris cells of step 5) the antibiotic resistance gene for zeocin.

6. The method according to claim 5, wherein the wild strain of P. pastoris is the strain PPS-9010.

7. The method according to any one of claims 5 to 6, wherein the plasmid of step 2) prior to its use in step 2), is previously digested with the restriction enzyme Pmel for at least 10 hours, preferably from 12 to 24 hours and / or at a temperature range from 30-43 °C.

8. The method according to any one of claims 5-7, wherein the plasmid of step 2) is purified with a gel extraction kit after the digestion.

9. The method according to any one of claims 5 to 8, wherein the transformation of step 2) is carried out by electroporation.

10. Use of the Pichia pastoris mutant according to any of the claims 1-3 or the Pichia pastoris mutant obtained from the method according to anyone of claims 5-9, for preparing protein-glutamine glutaminase and / or in the manufacturing of food.

11. Use of the Pichia pastoris mutant according to any of the claims 1-3 or the Pichia pastoris mutant obtained from the method according to anyone of claims 5-9, in a method for the production food or food products selected from the list consisting of, of plant-based analogues of milk, milk products, plant-based beverages, low-allergenic gluten products, gluten free products and vegetable drinks.

12. A protein-glutamine glutaminase characterised in that said protein comprises an amino acid sequence SEQ ID NO:1 or homologues thereof having an identity of at least 90%.

13. The protein-glutamine glutaminase according to claim 12, characterised because it comprises a histidine tag in the N terminal position and the absence of the signal peptide (21 aa).

14. The protein-glutamine glutaminase according to any one of the claims 12-13 for use as glutaminase.

15. A recombinant plasmid which comprises a DNA sequence, SEQ ID NO:

316. A Process to obtain the protein-glutamine glutaminase according to anyone of claims 12-14, characterized in that, the process comprises the use the Pichia pastoris mutant strain according to anyone of claims 1-3, or the Pichia pastoris mutant obtained from the method according to anyone of claims 5-9, as a fermentation strain.

17. The process to obtain protein-glutamine glutaminase according to claim 16 which comprises cultivating the Pichia pastoris mutant in a culture medium to produce glutaminase in the culture.

18. The process to protein-glutamine glutaminase according to anyone of claims 16-17, which comprises the following steps:a) culturing the fermentation strain Pichia pastoris mutant in a fermentation medium, b) adding methanol to the culture obtained in step a), andc) removing the cells from the fermentation medium obtained in step b), to obtain a supernatant comprising the targeted protein-glutamine glutaminase, and d) optionally providing the protein-glutamine glutaminase obtained of step c) as a powder by freeze drying or spray drying processes.

19. Use of the protein-glutamine glutaminase according to any one of the claims 12-15 or to the protein-glutamine glutaminase obtained according to any one of the claims 16-18, as food enzyme, preferably in the manufacturing of food products, plant-based beverages and vegetable drinks.

20. Use of the protein-glutamine glutaminase according to any one of the claims 12-15 or to the protein-glutamine glutaminase obtained according to any one of the claims 16-18, in a process for deaminating vegetable proteins, in particular to improve their proteins stability and or solubility in plant-based beverages.

21. A DNA sequence which encodes a protein according to any one of the claims 12-15, or to the protein-glutamine glutaminase obtained according to any one of the claims 16-18, which comprises the nucleotide sequence according to SEQ ID N:3.

22. The Pichia pastoris mutant yeast strain, strain characterized in that, the mutant carries a recombinant plasmid expressing protein-glutamine glutaminase, wherein the plasmid is a modification of the plasmid pPICZ, and it is characterised because it comprises:a plasmid backbone pBIZa 1.4,an AOX1 promoter region,a Zeocin antibiotic resistance gene,a Ost1-aF region, as an extracellular secretion signal andcontaining a DNA sequence SEQ NO:2a target product coding sequence protein-glutamine glutaminase, characterized because it comprises the amino acid sequence SEQ ID NO:1.

Images